Abstract

Mice are the experimental tool of choice for the majority of immunologists and the study of their immune responses has yielded tremendous insight into the workings of the human immune system. However, as 65 million years of evolution might suggest, there are significant differences. Here we outline known discrepancies in both innate and adaptive immunity, including: balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and λ5) and T cell (ZAP70 and common γ-chain) signaling pathway components, Thy-1, γδ T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression. We also provide examples, such as multiple sclerosis and delayed-type hypersensitivity, where complex multicomponent processes differ. Such differences should be taken into account when using mice as preclinical models of human disease.

Mice are the mainstay of in vivo immunological experimentation and in many respects they mirror human biology remarkably well. This conservation of function is reflected in recent reports on the sequencing of both the human and mice genomes, which reveal that to date only 300 or so genes appear to be unique to one species or the other (1). Despite this conservation there exist significant differences between mice and humans in immune system development, activation, and response to challenge, in both the innate and adaptive arms. Such differences should not be surprising as the two species diverged somewhere between 65 and 75 million years ago, differ hugely in both size and lifespan, and have evolved in quite different ecological niches where widely different pathogenic challenges need to be met—after all, most of us do not live with our heads a half-inch off the ground. However, because there are so many parallels there has been a tendency to ignore differences and in many cases, perhaps, make the assumption that what is true in mice—in vivo veritas—is necessarily true in humans. By making such assumptions we run the risk of overlooking aspects of human immunology that do not occur, or cannot be modeled, in mice. Included in this subset will be differences that may preclude a successful preclinical trial in mice becoming a successful clinical trial in human.

In this review our aim is not to suggest that the mouse is an invalid model system for human biology. Clearly, with so many paradigms that translate well between the species, and with the relative ease with which mice can now be genetically manipulated, mouse models will continue to provide important information for many years to come. Rather, our aim is to sound a word of caution. As therapies for human diseases become ever more sophisticated and specifically targeted, it becomes increasingly important to understand the potential limitations of extrapolating data from mice to humans. The literature is littered with examples of therapies that work well in mice but fail to provide similar efficacy in humans (2, 3, 4, 5, 6, 7). By focusing on some known differences between mouse and human immunology we hope to spur interest in this area and encourage others to note differences where they occur.

Structure and general characteristics

The overall structure of the immune system in mice and humans is quite similar. As this topic has been recently reviewed in depth (8), we will not go into great detail here. One difference worth noting is that whereas mice have significant bronchus-associated lymphoid tissue, this is largely absent in healthy humans (9), possibly reflecting a higher breathable Ag load for animals living so much closer to the ground.

The balance of lymphocytes and neutrophils in adult animals is quite different: human blood is neutrophil rich (50–70% neutrophils, 30–50% lymphocytes) whereas mouse blood has a strong preponderance of lymphocytes (75–90% lymphocytes, 10–25% neutrophils) (10). It is not clear what, if any, functional consequence this shift toward neutrophil-rich blood in humans has had.

Innate immunity

One of the first lines of defense in higher organisms, and often the only defense in lower animals, is the growing family of antimicrobial peptides, and in particular the defensins. These are important in mucosal defense in the gut and in epithelial defense in skin and elsewhere (12, 13). Neutrophils are a rich source of leukocyte defensins in humans, but defensins are not expressed by neutrophils in mice (14). In contrast, Paneth cells, which are present in the crypts of the small intestine, express >20 defensins (cryptdins) in mice but only two in human, likely reflecting different evolutionary pressures related to microorganism exposure through food intake. There are also differences in processing of defensins (Table I⇓⇓).

The last few years have seen a renewed focus on the field of innate immunology, spurred in large part by identification of the Toll-like family of receptors—the TLRs (15). This field is still relatively young and so far a limited number of differences have been noted between mice and humans (Table I⇑⇑).

There has been considerable controversy as to whether human macrophages express NO. Expression of functional inducible NO synthase (iNOS; NOS2) in mouse macrophages has been clearly demonstrated and iNOS mRNA is readily induced by IFN-γ and LPS (16). However, these same inflammatory mediators have failed to show consistent effects on human macrophages, hence the confusion. Recent work suggests that other mediators, such as IFN-αβ, IL-4 plus anti-CD23, and various chemokines, are actually far more efficient in inducing iNOS in human macrophages (17). However, the controversy is not dead yet (18).

Using different strains of mice a susceptibility locus for CMV infection, cmv1, was identified and later shown to encode the Ly49 family of proteins (19). There are at least 14 members and most are expressed on NK and NKT cells, where the majority act as NK inhibitory receptors for MHC I molecules. The Ly49 family is absent in humans, who use the KIR family as NK inhibitory receptors (20). KIR proteins are highly diverged from the Ly49 family and have Ig rather than C-type lectin domains in their extracellular domain; however, similarly to Ly49 they also recognize MHC class I. The ligands for mouse and human NKG2D differ: in humans, NKG2D binds the polymorphic MHC class I-like molecules MHC-I chain-related A, MHC-I chain-related B, and the UL16 binding protein family, whereas in mouse NKG2D binds to H-60 and Rae1β. The significance of these differences to CMV infection and to NK biology in general have not been determined.

Adaptive immunity

FcR represent a link between the adaptive immune system, which generates Ab, and the innate immune system, which can respond to Ab-Ag complexes through capture by FcR expressed on macrophages, neutrophils, eosinophils, mast cells, and dendritic cells. There are several differences in FcR expression between mice and humans. In humans, FcαRI (CD89) is an important IgA receptor expressed by neutrophils, eosinophils, monocytes/macrophages, dendritic cells, and Kupffer cells (21). Mice lack FcαRI and presumably use alternative receptors, such as Fcα/μR, the transferrin receptor (CD71) and polymeric IgR, which also binds IgM. Humans also express two IgG receptors not found in mice: FcγRIIA and FcγRIIC are closely related single-chain FcR, each of which has a single ITAM motif in the intracellular domain. In contrast, most other FcR associate with ITAM-containing signal transduction subunits (22).

In addition to differences in FcR there are well-known differences in expression of Ig isotypes between mice and humans, and direct correlations between subtypes within classes in each species are hard to make. Mice make IgA, IgD, IgE, IgM, and four subtypes of IgG: IgG1, IgG2a, IgG2b, and IgG3. Interestingly, in the C57BL/6, C57BL/10, SJL, and NOD strains of mice there is no expression of IgG2a, instead these mice express the novel IgG2c (23). Humans in contrast express two subtypes of IgA—IgA1 and IgA2—along with single forms of IgD, IgE, and IgM. In humans there are also four subtypes of IgG: IgG1, IgG2, IgG3, and IgG4; however, these are not direct homologues of the mouse proteins. While different subtypes have differing abilities to bind FcR or fix complement, the differences between mice and humans are not considered significant. In contrast, there are differences in class switching: in mice, IL-4 induces IgG1 and IgE, whereas in humans, IL-4 induces switching to IgG4 and IgE. In contrast, IL-13 has no effect on mouse B cells but induces switching to IgE in humans (24).

There are some interesting differences in B cell development that relate to the roles of several signaling molecules. BLNK (Src homology-2 domain containing leukocyte-specific phosphoprotein-65) is an adapter protein that is rapidly phosphorylated by Syk after cross-linking of the B cell Ag receptor. It then serves as a scaffold for downstream signaling components such as Grb2, Vav, Nck, and PLC-γ. B cell development in mice lacking BLNK is blocked at the pro-B to pre-B transition, resulting in low numbers of IgM+ B cells, but no mature IgMlowIgDhigh B cells, appearing in the periphery (25). A naturally occurring mutation in the human BLNK protein has been identified that results in a splicing defect preventing protein expression. In this patient there was also a block in the pro-B to pre-B transition; however, there was also a complete absence of B cells in the periphery, suggesting a more severe block in human B cell development than in mice (26).

Similarly discrepant phenotypes have been noted in mice lacking functional BCR-associated tyrosine kinase Btk (27) and in mice lacking λ5 (28), the L chain component of the pre-BCR (Table I⇑⇑). Differences in mature B cells between mice and humans were recently reviewed (29), and include mutually exclusive expression of CD5 and CD23 on mouse but not human B cell subsets, and CD38 expression on human, but not mouse, plasma cells.

The discrepant phenotypes discussed above for BLNK, Btk, and λ5 should be treated with some caution as the human diseases usually arise due to mutations in the relevant genes rather than deletions of whole exons as seen in the mouse knockout models. In some cases, however, identical mutations have been found, or created, in mice and the discrepant phenotype remains. This is the case for human XLA and mouse XID, which both involve Btk (30, 31).

The development and regulation of T cells also differs between mice and humans. Thy-1 is a GPI-linked Ig superfamily molecule of unknown function. It is expressed on thymocytes and peripheral T cells in mice and has been widely used as a T cell marker in the thymus. In humans, however, it is only expressed on neurons. The basis of this tissue specificity is suggested to be the presence or absence of an Ets-1 binding site in the third intron of the gene (32).

Similar to the development of B cells, mutation of key signaling molecules in T cells has markedly different effects in mice and humans. Several cytokine receptors, including those for IL-2, IL-4, IL-7, IL-9, and IL-15, share a common signaling chain called common γ chain (γc). Perhaps not surprisingly, deletion or mutation of this gene, which is on the X chromosome, results in severe immunological defects. Interestingly, these differ between human and mouse XSCID (33, 34). Numerous mutations have been identified in the human γc gene that inhibit function, and in most of these cases the result is a dramatic decrease in the number of T cells and NK cells. However, B cell development is normal, although function is impaired, likely due to the lack of T cell help. In marked contrast, B cell numbers are greatly diminished in γc-null mice. Given that IL-7R deficiency in mice blocks both T and B cell development (35), but only blocks T cell development in humans (36), it is likely that B cell development in humans is independent of IL-7. The major signal transducer for γc is JAK3 and mutation of this gene phenocopies the γc mutation in both mice and humans; that is, a lack of T and NK cells in human with the addition of a severe B cell defect in mice (31).

Interesting differences have also been noted in ZAP70-deficient mice and humans. ZAP70 is essential for TCR signaling in both developing and mature T cells, and compromised signaling results in SCID. In humans the defect results in normal numbers of CD4+ T cells and absent CD8+ T cells. However, the CD4+ T cells are nonfunctional. In contrast, an identical mutation introduced into the mouse ZAP70 results in a block in differentiation of both T cell subsets at the double-positive stage (37). It has been suggested that the “leakiness” of the human mutant is due to incomplete down-regulation of the protein tyrosine kinase Syk in human thymocytes, compared with mouse thymocytes (38).

The study of γ/δ T cells has revealed a number of significant differences between mice and humans. T cells expressing γ/δ TCR are found in all organisms that have α/β receptors and yet their function is still largely an enigma (39). Mouse skin contains a large fraction of cells bearing a TCR encoded by a single Vγ and Vδ gene. These Vγ5-Vδ1 T cells appear to be oligoclonal, reside in the epidermis, and are known as dendritic epidermal T cells (DETC). DETC represent the predominant T cell in mouse skin, whereas cells bearing α/β receptors predominate in human skin and are found mostly in the dermis. Indeed, a cell with DETC characteristics has not been identified in humans (40). Human but not mouse γ/δ T cells have been suggested to recognize Ag presented by CD1 molecules—in particular CD1b (41). Interestingly, of the five CD1 molecules found in humans (designated CD1a, b, c, d, and e), only CD1d is expressed in mice (41). Similarly to γ/δ T cells the CD1 family of molecules has been implicated in the pathogenesis of tuberculosis, but their precise role has yet to be defined (42, 43). The differing expression of CD1 genes between mice and humans may well turn out to impact activation of both α/β and γ/δ T cells in tuberculosis, as both subsets can recognize a variety of Ags presented by CD1 molecules.

An often critical component of adaptive immunity is the skewing of T cell differentiation toward Th1 or Th2 phenotypes and this process represents another area of interaction between the innate and adaptive arms of immunity. In humans, the type I IFN, IFN-α, is secreted by several cell types in response to viral infection, including macrophages, and acts on T cells to induce Th1 development. This process is dependent upon STAT4 activation, and its recruitment to the IFN-α receptor by STAT2. In mice, however, IFN-α fails to induce Th1 cells and does not activate STAT4 (44).

The existence of polarized T cell populations was first demonstrated by Mosmann and colleagues (45) and since then has become a guiding principle for T cell activation. While polarization is relatively easy to observe in mice the paradigm has never been as clear-cut in the human system. Th1 and Th2 cells can certainly be found in human disease (46, 47); however, there is a growing recognition that in many diseases clear distinctions cannot be made and that T cells of both persuasions can often be generated simultaneously (48, 49, 50). For example, in mice, IL-10 is considered to be a Th2 cytokine, whereas in humans both Th1 and Th2 cells can make IL-10 (51). The response of mice and humans to schistosomiasis is remarkably different. Epidemiological data suggest that a Th2 response involving eosinophils and IgE may be key to combating infection in humans (52), whereas in mice effector cell activation by IFN-γ, a Th1 response, is essential for clearance of the parasite (53).

To become fully activated T cells require both a primary, Ag-dependent signal, and a second, Ag-independent or costimulatory signal. One of the best characterized costimulatory receptors is CD28, which is expressed by close to 100% of mouse CD4+ and CD8+ T cells. In contrast, only 80% of human CD4+ and 50% of human CD8+ T cells express CD28 (54), perhaps accounting for the remarkable efficacy of CTLA-4Ig in blocking T cell activation in mice. It will be interesting to see if expression of the CD28-related costimulatory molecule ICOS segregates with CD28− T cells in humans. The recent report on the identification of a human ICOS deficiency pointed to a further difference between costimulation in mice and humans. Whereas in mice the loss of ICOS does not affect either the number of mature B cells, their maturation status or their secretion of IgM (55, 56), the loss of ICOS in humans results in a severe reduction in B cell number, maturation status and secretion of IgM (57). Given the critical role of T cell CD40L in T-B interactions it would be interesting to know what the level of CD40L expression was on this patient’s T cells and whether expression of this molecule is dependent upon ICOS signaling in humans. Two novel members of the B7 family of costimulatory molecules, B7-H3 and DC-SIGN, have recently also been suggested to have different roles in mice and humans (Table I⇑⇑).

P-selectin is constitutively expressed by endothelial cells (EC) and mediates leukocyte rolling by interactions with specific sugar residues carried by mucins. Interestingly, murine P-selectin can be strongly up-regulated by inflammatory mediators such as TNF and LPS, whereas the human gene is nonresponsive (58). It is interesting to speculate as to whether E-selectin in humans, which is strongly up-regulated by TNF, is the more important selectin on human EC for mediating leukocyte rolling.

Once activated, human T cells express MHC class II molecules whereas murine T cells do not. It has been suggested that human T cells can capture, process, and present Ag and that they express B7 and may therefore help to amplify an ongoing immune response (59, 60). In contrast, Ag presentation by T cells may also promote T cell anergy (61) or activation-induced cell death. It is not clear why this function is nonessential in mice, but it is an attractive hypothesis that it may relate to T cell homeostasis and the requirement in humans for maintaining, in a limited compartment, a greater diversity of memory T cells for a considerably longer period of time than is required in mice. T cell homeostasis requires programmed cell death (apoptosis) of unwanted cells. Caspase 8 and caspase 10 are downstream of death receptors in humans and overlap in some of their functions (62). Mice lack caspase 10 and the deletion of caspase 8 is embryonic lethal. Lack of caspase 8 in humans results in immunodeficiency, suggesting a role for this effector in lymphocyte activation as well as death (63). Greater redundancy in death receptor regulators in humans may relate to the longer lifespan and associated increased risk of developing cancer.

A critical step in activation of a T cell is the generation of a sustained calcium flux. In human T cells the inward flow of calcium ions is balance by an outward flow of K+, mediated in large part by the Kv1.3 K+ channel. Inhibitors of this channel very specifically block T cell activation in vitro and are being pursued as novel immunosuppressive agents (64). However, in vivo evidence to support such a function is missing as mouse T cells do not express this channel (65).

The movement of immune cells into and through tissues is coordinated by a huge array of chemokines and chemokine receptors and, not surprisingly, differences have emerged between the murine and human systems. While it is still too early to say definitively what such differences may mean, as there appears to be considerable redundancy built into the system, it is worth noting what is currently known. CXCR1 is present in humans but not in mice (66). The chemokines IL-8 (CXCL8), neutrophil-activating peptide-2 (CXCL7), IFN-inducible T cell α-chemoattractant (CXCL11), monocyte chemoattractant protein (MCP)-4 (CCL13), HCC-1 (CCL14), hemofiltrate CC chemokines-2 (CCL15), pulmonary and activation-regulated chemokine (CCL18), myeloid progenitor inhibitory factor-1 (CCL23), and eotaxin-2/3 (CCL24/CCL26) have all been identified in humans but not in mice. Conversely, CCL6, CCL9, lungkine (CXCL15), and MCP-5 (CCL12) have been identified in mice but not humans (66, 67).

Differences in immune system biology

Multiple sclerosis (MS) provides a fine example of both differences and similarities between mouse and human immunology. MS is a multifactorial disease that appears to have a large autoimmune component (68). Experimental autoimmune (allergic) encephalomyelitis is a widely used model for MS that mimics the demyelination seen in central and peripheral nerves in MS. Several studies have indicated that IFN-γ is protective in experimental autoimmune (allergic) encephalomyelitis as neutralizing Abs exacerbate disease, potentially by blocking induction/activation of suppressor activity (69, 70). It was surprising, therefore that clinical trials were not successful; indeed they were stopped because treatment with IFN-γ was found to exacerbate disease (4). In contrast, studies in mice suggested that blocking VLA-4 (α4β1 integrin)-VCAM-1 interaction might help in MS (71) and this has indeed carried through successfully into human trials (72). These studies highlight how caution is required when extrapolating results from mouse studies to the clinic, but suggest that mouse models can successfully predict some therapies for human disease.

An interesting difference exists in the appearance of delayed-type hypersensitivity (DTH) reactions in mice and humans. In humans, around four hours after Ag challenge neutrophils can be seen forming a “cuff” around the venules. This is followed by a dramatic influx of mononuclear cells, such that by 24–48 h the lesion is mostly mononuclear with a mix of T cells and macrophages (73). Paradoxically, in mice where the peripheral blood has a relative paucity of neutrophils compared with humans, the DTH response tends to be more neutrophil rich (74). In addition, elicitation of murine DTH requires much higher concentrations of Ag than in humans.

There is now considerable evidence that human EC can present Ag to resting memory CD4+ and CD8+ T cells (75, 76, 77), whereas in mice, CD8+ T cells can be activated by EC (78), but CD4+ T cells cannot (B. Rosengard, personal communiction). As CD4+ T cell-mediated activation of macrophages is thought to drive human DTH responses the suggestion has arisen that in humans, Ag transport to lymph nodes by Langerhans cells may not be necessary as EC may trigger the recall response at the site of challenge. A teleological argument can be made for the need to present Ag locally in humans but not necessarily in mice. It has been estimated that once a cell enters the lymphatics in humans it takes ∼24 h to return to the circulation if it is not retained in a node (79). Based on the higher cardiac output of mice as a proportion of their total blood volume compared with humans (5–10 ml/min, 2 ml total volume in mice; 5 L/min, 5 liter total volume in humans) it is reasonable to suppose that return of lymph is at least as fast in mice as it is in humans. Then it becomes a matter of scale. We calculate that an Ag traveling from toe to an inguinal lymph node in the groin should take ∼12 h in humans and 20 min in mice. As the human DTH response begins around 4 h after secondary Ag challenge, it is possible that triggering of recall responses may occur by different mechanisms in mice and humans, involving draining of Ag to lymph nodes in mice, compared with local Ag presentation in humans.

Both human and mouse EC express MHC class I. Most human EC in vivo also constitutively express MHC class II molecules, whereas mouse EC do not (80). Thus, human EC can present Ag to CD4+ T cells, as well as to CD8+ T cells. A major costimulatory molecule on human EC is CD58 (LFA-3), a ligand for CD2 (81). Mice do not have the gene for CD58, which arose by CD2 gene duplication after the two lineages split. In mice the CD2 ligand is CD48; however, the distribution of this molecule differs from that of CD58 in humans, and the two-dimensional affinity for the mouse CD2-CD48 interaction is 40- to 50-fold lower than that for human CD2-CD58 interactions (82). In addition, gene deletion and Ab blocking studies have shown that mouse T cell activation is much less dependent on CD2 interactions than is the case for human T cells. Human EC also express CD40 and the ICOS ligand GL-50, whereas murine EC do not (83, 84).

The Ag presenting ability of human EC may have significant consequences for transplantation. For example, in many rodent models vascularized grafts are tolerizing, whereas such grafts are rapidly rejected in humans (5). Numerous studies have shown that purging mouse tissues of CD45+ cells before transplantation dramatically extends the life of the graft, sometimes even inducing tolerance. In sharp contrast, purging human tissues of CD45+ cells provides no benefit as the grafts are still rapidly rejected (6). In addition, the establishment of microchimerism in mice has been quite successful in inducing tolerance, whereas this has not been the case in humans (7). The implication of these findings is that there are major differences between mice and humans in their responses to grafted tissue, and that this may relate to the Ag-presenting ability of human, but not murine, EC.

Natural selection and the immune system

Most, if not all, of the differences we have noted between mouse and human immunology have likely become fixed during the 65 million years since our divergence because they provide some selective advantage. In all likelihood these adaptations are in response to new pathologic challenges from microorganisms, which have very short generation times and often have high mutation rates (85). In consequence, mammalian MHC molecules and NK cell inhibitory receptors have also evolved rapidly (9, 86). It should also be noted that some changes may be fixed primarily as a result of the nonimmune role of that gene—reiterative use of genes is a well recognized phenomenon during development, a good example being the important nonimmunological role of VCAM in chorioallantoic fusion and placentation (87). Thus, both the immune system as a whole, and some of its individual components (B and T cell repertoires) are shaped by natural selection.

Mice evolved in a quite different environment to humans and have been exposed to different Ags and their immune systems might therefore be expected to have evolved in subtly different ways. Mice not only live in different ecological niches, they are also much smaller and have significantly shorter lifespans. These are not trivial differences—as noted above, leukocyte transit times may be quite different in mice and humans, and a larger, broader repertoire of B and T cells must be maintained for many years in humans (up to 50 mouse lifetimes). Thus many changes may be to accommodate increased size of the organism, to regulate larger and more diverse pools of Ag-specific cells, and to provide greater checks and balances to combat the increased somatic mutation load that longer-lived animals necessarily carry.

Summary

While it is hard to draw global conclusions about the significance of differences between mouse and human immunology, it is worth considering the possibility that any given response in a mouse may not occur in precisely the same way in humans. While caution in interpreting preclinical data obtained in mice is clearly warranted, we believe that with these caveats in mind, mice will continue to be the premiere in vivo model for human immunology and will be absolutely essential for continued progress in our understanding of immune system function in health and disease.

Acknowledgments

Due to space limitations we are unable to cite as many primary references as we would wish and so have instead steered the reader toward many excellent reviews. We offer apologies to those colleagues whose work we were unable to include. We thank the following people for helpful suggestions: Jeffrey Bender, Mark Boothby, Alfred Bothwell, Michael Cahalan, David Camerini, Paolo Casali, George Chandy, Cheong Chang, Nick Crispe, Olja Finn, David Fruman, Geoff Kansas, Mitch Kronenberg, Tom Lane, Klaus Ley, Ruslan Medzhitov, Andre Ouellette, Peter Parham, Howard Petrie, Jordan Pober, Bruce Rosengard, Michael Selsted, Carl Ware, Marian Waterman, and Arthur Weiss. We also thank David Fruman and Craig Walsh for critical reading of the manuscript.